The present invention relates to an apparatus for investigating the kinetics of fast chemical reactions in solution using the temperature-jump-relaxation method with spectrophotometric observation of the time course of the reaction with special attention to the fluorimetric technique.
In conventional temperature-jump measurements a sample solution comprising a chemical system under investigation is filled into an optical sample cell having a volume of a few cubic centimeters. The cell is placed in the absorption light path of a spectrophotometer. The equilibrium parameters of the chemical system are changed by a stepwise increase of the temperature ("temperature jump"). Characteristic changes of the absorption spectrum after the temperature-jump indicate how fast the chemical system attains a new equilibrium state and the extent of the concentration changes of the reactants and reaction products. Usually, the temperature-jump is produced by discharging a high-voltage capacitor charge through the electrically conducting sample. To permit such a discharge, the sample cell has two electrodes made of noble metal or stainless steel forming a gap which is perpendicular to the light path. The discharge time and thus the heating time are of the order of one microsecond. The temperature change is several degrees centrigrade. (C. F. M. Eigen et al. in: Zeitschr. f. Elektrochemie, Vol. 62, p. 652 (1959), M. Eigen and L. De Maeyer in: Technique of Organic Chemistry, Ed. A. Weissberger, Vol. 8/II, p. 395, Wiley, N.Y. 1963.) Known modifications of the temperature-jump technique use either the cable discharge method or heating by a microwave or infrared-laser pulse.
At low concentrations, and thus low optical absorption, the measurement of the concentration changes by means of absorption becomes difficult. On the other hand, the concentrations cannot be chosen arbitrarily with respect to the equilibrium constant of the chemical system under investigation. If the equilibrium constant of a first order reaction is very large, a temperature-jump experiment only leads to a significant displacement of the equilibrium at low concentrations. This is especially true for many biochemical reactions where the substances involved are active at extremely low concentrations. Furthermore, the costs of material preparation are of importance in biochemical studies. Thus it is of special interest to perform measurements using very small samples and low concentrations. It is also important to improve the specificity of the spectrophotometric detection method.
In the case of static spectrophotometers an improved high sensitivity at low concentrations as well as a high specificity can be achieved by measuring the fluorescence light which is emitted by many organic molecules when excited by light of shorter wavelength, especially ultraviolet light.
Temperature-jump measurements using fluorescence detection involve extremely difficult problems. The signal-to-noise-ratio is proportional to the square root of the light intensity times the signal risetime. The signal risetime of static spectrophotometers is of the order of one second. For microsecond temperature-jump measurements of small differential effects the light intensity should be 10.sup.6 times higher than with static measurements. The most obvious way to obtain a higher light intensity would be to use extremely powerful high-pressure lamps and monochromators. This possibility, however, is limited for chemical reasons, because of the finite photochemical stability of the sample, and for physical reasons, because the highest light intensities cannot be obtained without a decrease in light stability which is also very important for a high-resolution instrument. Due to these difficulties temperature-jump measurements using fluorimetric detection have only been successful in a few cases so far. At low concentrations the signal was lost in the noise.